In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion prot...In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.展开更多
The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene...The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.展开更多
[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the ...[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.展开更多
目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙...目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙基-β-D-硫代半乳糖苷(IPTG)对重组质粒进行诱导表达,确定各重组蛋白的最佳参数。分别用AR内源性抗体和GST标签抗体进行Western blot鉴定,分析诱导前、诱导后细菌裂解液中的目的蛋白,并利用谷胱甘肽琼脂糖树脂进一步纯化。将纯化后蛋白与病毒荧光探针进行孵育,复合物于非变性凝胶中电泳检测纯化蛋白功能。结果成功构建了AR全长和3个功能域截短体的原核表达质粒。PCR和双酶切鉴定均显示在各插入片段大小相符处出现阳性条带,且测序结果与NCBI GenBank标准株比对一致。其中,96 ku GST-AR-NTD+DBD与86 ku GST-AR-NTD两个融合蛋白得以成功表达及后续纯化。纯化蛋白可与病毒基因组DNA直接结合。结论来源同一基因序列的AR截短体重组质粒原核表达条件不同,纯化后的AR蛋白可用于深入了解AR各功能域与其他分子间的直接相互作用机制。展开更多
文摘In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
文摘The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.
基金Supported by Project of Science and Technology Development of Jilin Province(20140204018YY)
文摘[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.
文摘目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙基-β-D-硫代半乳糖苷(IPTG)对重组质粒进行诱导表达,确定各重组蛋白的最佳参数。分别用AR内源性抗体和GST标签抗体进行Western blot鉴定,分析诱导前、诱导后细菌裂解液中的目的蛋白,并利用谷胱甘肽琼脂糖树脂进一步纯化。将纯化后蛋白与病毒荧光探针进行孵育,复合物于非变性凝胶中电泳检测纯化蛋白功能。结果成功构建了AR全长和3个功能域截短体的原核表达质粒。PCR和双酶切鉴定均显示在各插入片段大小相符处出现阳性条带,且测序结果与NCBI GenBank标准株比对一致。其中,96 ku GST-AR-NTD+DBD与86 ku GST-AR-NTD两个融合蛋白得以成功表达及后续纯化。纯化蛋白可与病毒基因组DNA直接结合。结论来源同一基因序列的AR截短体重组质粒原核表达条件不同,纯化后的AR蛋白可用于深入了解AR各功能域与其他分子间的直接相互作用机制。
